Fluorescence quenching azo dyes, their methods of preparation and use

ABSTRACT

Disclosed is a group of azo quencher compositions useful as fluorescence quenchers having the general structure of formula 1, methods of making or using the compositions, and kits comprising the composition.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 12/252,721filed Oct. 16, 2008, which is a divisional of U.S. application Ser. No.10/987,608 filed Nov. 12, 2004, now U.S. Pat. No. 7,439,341, whichclaims priority benefits to U.S. Provisional Application No. 60/520,077filed Nov. 14, 2003. These applications are incorporated herein byreference in their entirety.

FIELD OF THE INVENTION

This invention pertains to compositions that are capable of quenchingthe fluorescence of fluorophores and to methods for making and usingthem. The invention also provides kits that contain at least one of thedisclosed quencher compositions.

BACKGROUND OF THE INVENTION

Light quenching processes that rely on the interaction of two dyes astheir spatial relationship changes can be used in convenient processesfor detecting and/or identifying nucleotide sequences and otherbiological phenomena. In one such method the change in fluorescence of afluorescent donor or quencher can be monitored as two oligonucleotides(one containing a donor and one containing a quencher) bind to eachother through hybridization. The binding can be detected withoutintervening purification steps that separate unhybridized fromhybridized oligonucleotides.

Another method for detecting hybridization using fluorophores andquenchers is to link fluorescent donors and quenchers to a singleoligonucleotide such that there is a detectable difference influorescence when the oligonucleotide is unhybridized as compared towhen it is hybridized to its complementary sequence. For example, apartially self-complementary oligonucleotide designed to form a hairpincan be labeled with a fluorescent donor at one end and a quencher at theother end. Intramolecular annealing into the hairpin form can bring thedonor and quencher into sufficient proximity for fluorescent quenchingto occur. Intermolecular annealing of such an oligonucleotide to atarget sequence disrupts the hairpin, which increases the distancebetween the donor and quencher and results in an increase in thefluorescent signal of the donor.

However, oligonucleotides are not required to have hairpins for thislater method to work efficiently. The fluorophore and quencher can beplaced on an oligonucleotide such that when it is unhybridized and in arandom coil conformation, the quencher is able to quench fluorescencefrom the fluorophore. Once the oligonucleotide hybridizes to acomplementary nucleotide sequence it becomes more extended and thedistance between the fluorophore and quencher is increased, resulting inincreased fluorescence.

Oligonucleotides labeled in a similar manner can also be used to monitorthe kinetics of PCR amplification. In one version of this method theoligonucleotides are designed to hybridize to the 3′ side (“downstream”)of an amplification primer so that the 5′-3′ exonuclease activity of apolymerase digests the 5′ end of the probe, cleaving off one of thedyes. The fluorescence intensity of the sample increases and can bemonitored as the probe is digested during the course of amplification.

Similar oligonucleotide compositions find use in othermolecular/cellular biology and diagnostic assays, such as in end-pointPCR, in situ hybridizations, in vivo DNA and RNA species detection,single nucleotide polymorphism (SNPs) analysis, enzyme assays, and invivo and in vitro whole cell assays.

Perhaps the most common mechanism of fluorescent quenching is known asFRET (fluorescent resonance energy transfer). For FRET to occur afluorescent donor and a fluorescent quencher must be within a suitabledistance for the quencher to absorb energy from the donor. In addition,there must be overlap between the emission spectrum of the fluorescentdonor and the absorbance spectrum of the quencher. This requirementcomplicates the design of probes that utilize FRET because not allpotential quencher/donor pairs can be used. For example, the quencherknown as BHQ-1, which absorbs light in the wavelength range of about520-550 nm, can quench the fluorescent light emitted from thefluorophore, fluorescein, which fluoresces maximally at about 520 nm. Incontrast, the quencher BHQ-3, which absorbs light in the wavelengthrange of about 650-700 nm would be almost completely ineffective atquenching the fluorescence of fluorescein through FRET but would bequite effective at quenching the fluorescence of the fluorophore knownas Cy5 which fluoresces at about 670 nm. In general, the number ofquenchers known that are capable of quenching the fluorescence of anygiven fluorophore is quite limited. For example with fluorescein, only alimited number of suitable quenchers are known and they are quiteexpensive to purchase commercially. Because fluorescein is one of themost commonly used fluorophores, new quenchers that can quenchfluorescent light in the 520 nm range of fluorescein are needed.Similarly, quenchers for other known fluorophores are also needed.

Ideally, new quenchers will not fluoresce so that backgroundfluorescence is minimized. This will allow for an increased signal tonoise ratio in the probes that contain them, resulting in more sensitiveprobes. In addition, the lack of a secondary fluorescence facilitatesthe use of additional fluorophores in multiplexed assay formats whichutilize multiple distinct probes each containing a differentfluorophore. If a quencher emitted light in a region, then additionalprobes could not bear fluorophores that emit light in that region.

New quenchers should also have physical properties that facilitate theirpurification and the purification of probes into which they areincorporated. They should also be chemically stable so that they can beincorporated into biological probes and used in assays withoutsignificant degradation. The quenchers should contain suitable reactivemoieties to provide for their convenient incorporation into biologicallyrelevant compounds such as lipids, nucleic acids, polypeptides, and morespecifically antigens, steroids, vitamins, drugs, haptens, metabolites,toxins, environmental pollutants, amino acids, peptides, proteins,nucleotides, oligonucleotides, polynucleotides, carbohydrates, and thelike. Lastly, the most useful compositions should be easilymanufactured.

The invention provides nonfluorescing, fluorescence-quenchingcompositions, some of which have strong fluorescence quenchingproperties in the 520 nm range. Moreover, the quenchers of the presentinvention are chemically stable and can be easily manufactured andpurified. The compositions can be incorporated into biologicallyrelevant compounds and, in many cases, impart useful purificationproperties to these compounds. These and other advantages of theinvention, as well as additional inventive features, will be apparentfrom the description of the invention provided herein.

BRIEF SUMMARY OF THE INVENTION

The invention provides a novel group of azo quencher compositions thatare useful as quenchers of fluorescence and to methods for making andusing them. The quenchers of this invention are termed dark quenchersbecause they release the energy they absorb from fluorophores withoutgiving off light. The quenchers contain an azo bond which joins R₇ to aconjugated ring system. The quenchers have the general formula shownbelow in Formula 1.

In Formula 1, R₁₋₆ can individually be electron withdrawing groups suchas halogen, NO₂, SO₃R, SO₂N(R)₂, CN, NCS, keto, alkoxy groups, or C₁-C₁₀alkyl groups, aryl groups, or heteroaryl groups. In addition, the R₁/R₂pair, R₃/R₄ pair, R₄/R₅ pair and R₅/R₆ pairs can be combined to formring structures having five or six ring members. These ring structurescan be substituted. R₇ can be any aryl group that can be joined to theconjugated ring system by an azo bond to form a compound that is capableof quenching the fluorescence of a fluorophore. The quenchers can bederivatized to facilitate their conjugation to a variety of biologicallyrelevant compounds, including lipids, nucleic acids, peptides, proteins,and the like. The invention also provides kits comprising, in one ormore containers, at least one quencher dye composition of the presentinvention, and instructions for using that composition.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the relative fluorescence collected during PCR reactionswhich contained three different probes (A-C). A is SEQ ID NO. 4(AzoQuencher)-ACCCGTTCACCCTCCCCCAGT-(6-FAM); B is for SEQ ID NO. 5(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(AzoQuencher); and C is for SEQ ID NO. 6(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(BHQ2).

FIG. 2 shows the absorbance spectrum of an oligonucleotide SEQ ID NO. 10having the composition (AzoQuencher)-CAGAGTACCTGA

DETAILED DESCRIPTION OF THE INVENTION

The invention provides a novel group of azo quencher compositions thatare useful as quenchers of fluorescence and to methods for making andusing them. The quenchers of this invention release the energy theyabsorb from fluorophores without giving off light. Consequently, theyare called dark quenchers. The quenchers contain an azo bond and havethe general formula shown below in Formula 1.

In Formula 1, R₁₋₆ can individually be electron withdrawing groups suchas halogen, NO₂, SO₃R, SO₂N(R)₂, CN, NCS, keto, alkoxy groups, or C₁-C₁₀alkyl groups, aryl groups, or heteroaryl groups. In addition, the R₁/R₂pair, R₃/R₄ pair, R₄/R₅ pair and R₅/R₆ pairs can be combined to formring structures having five or six ring members. These ring structurescan be substituted. In certain embodiments one or more of R₁₋₆ can behydrogen.

In Formula 1, R₇ can be any aryl group that can be joined to theconjugated ring system by an azo bond to form a compound that is capableof quenching the fluorescence of a fluorophore. Suitable aryl groupsinclude phenyl, naphthyl, benzyl, xylyl, tolyl, pyridyl, and anilyl,among other groups. Preferably, the aryl groups are substituted orderivatized with at least one linking group for linking the quenchermolecule to other molecules of interest.

In one preferred embodiment, R₇ is the compound of Formula 2, whereinthe aryl ring is an aniline which can be substituted with various groupsat positions L and L′.

L and L′ can be either nonreactive groups or reactive linking groups.For example, in one embodiment one of L or L′ can be a nonreactive groupsuch as an alkyl group, preferably an ethyl group, and the other can bea hydroxyethyl group which can be modified further to facilitate linkingthe quencher to other molecules of interest. Alternatively, both L andL′ can be hydroxyethyl groups, as shown in Formula 3, below, either orboth of which can be modified further for linking. One of skill in theart would recognize that hydroxy alkyl chains of any length or otherlinking groups, as described in more detail below, could be used tomodify the aniline.

In Formula 3, the R₈ and R₉ groups can be any of a variety of linkinggroups which can be tailored for use. For example, one or both of R₈ andR₉ can be phosphoramidite groups such as diisopropylamino cyanoethylphosphoramidite. Such a group would allow the quencher to react andbecome covalently attached to nucleophilic groups, particularly hydroxylgroups. In addition, one of R₈ or R₉ can be a hydroxyl protecting groupsuch as a silyl group or a trityl group such as a mono, di, ortri-methoxytrityl group. More preferably, one of R₈ and R₉ is a tritylgroup and the other is a phosphoramidite group. In a preferredembodiment of Formula 1, the azo quencher composition has the structureof Formula 4 wherein one of R₈ or R₉ is a trityl group and the other isa phosphoramidite.

In one embodiment, the quencher is a compound of Formula 13:

wherein CEP is cyanoethylphosphoramidite, and C₁₋₁₀ is a C₁ to C₁₀ alkylgroup. In another embodiment, the quencher is a compound of Formula 14:

wherein trityl is an alkoxytrityl group, CEP iscyanoethylphosphoramidite, and C₁₋₁₀ is a C₁ to C₁₀ alkyl group.

The invention also contemplates methods for preparing the disclosedcompositions. The reaction between the conjugated ring system and R₇precursors can be carried out by treating a suitable conjugated ringprecursor composition with a suitable nitrite, such as NaNO₂, or asuitable organic nitrite in a suitable solvent and subsequently withLiBF₄ to create a diazonium salt. The diazonium salt is then reactedwith the R₇ precursor, an aryl composition, to generate an azo bondedring system product. Methods for carrying out this reaction sequence aredescribed in more detail in Examples 1 and 2.

Suitable conjugated ring precursor compounds have a primary amine andhave the general structure of Formula 5. Specific embodiments of Formula5 include the structures of Formulas 6-8.

The azo quencher of Formula 1 can be further modified to facilitate itsuse. For example, reactive groups, such as amino, hydroxyl, and carboxylgroups on R₅ can be attached to linking groups or other molecules ofinterest. In one embodiment of Formula 4, R₈ can be reacted with atrityl group and R₉ can be reacted with N,N-diisopropylamino cyanoethylphosphonamidic chloride, as described in Example 2, to generate aphosphoramidite reagent suitable for reaction with a variety ofnucleophiles, especially including hydroxyl groups.

The phosphoramidite quenchers are ideally suited for incorporation intooligonucleotides. The phosphoramidite can be used to derivatize a solidsupport, as in Example 2, and the derivatized support can serve as thefoundation for oligonucleotide synthesis by standard methods. AlthoughExample 2 demonstrates the attachment of an azo-quencher compound tocontrolled pore glass, the method is more generally applicable to theattachment of the quencher to any solid support that contains a suitablenumber of free reactive nucleophilic groups, including polystyrene andpolypropylene. The solid support-bound azo-quencher andtrityl-protected, phosphoramidite quencher can both be used convenientlyin conjunction with automated oligonucleotide synthesizers to directlyincorporate the quencher into oligonucleotides during their chemicalsynthesis. In addition, the disclosed quenchers can be incorporated intooligonucleotides post synthetically. Such precursors and theoligonucleotides prepared with them are also contemplated by the presentinvention.

The disclosed quenching compositions can be linked to a variety ofuseful compounds other than oligonucleotides, provided that suitablereactive groups are present on those compounds. Such compounds includeantigens, antibodies, steroids, vitamins, drugs, haptens, metabolites,toxins, environmental pollutants, amino acids, proteins, carbohydrates,lipids, and the like.

For purposes of this invention the term “linking group” refers to achemical group that is capable of reacting with a “complementaryfunctionality” of a reagent, e.g., to the hydroxyl group of a nucleicacid, and forming a linkage that connects the azo quenching compound ofFormula 1 to the reagent. When the complementary functionality is anamine, preferred linking groups include such groups as isothiocyanate,sulfonylchloride, 4,6-dichlorotriazinyl, carboxylate, succinimidylester, other active carboxylate, e.g., —C(O)halogen, —C(O)OC₁₋₄ alkyl,or —C(O)OC(O)C₁₋₄ alkyl, amine, lower alkylcarboxy or—(CH₂)_(m)N⁺(CH₃)₂(CH₂)_(m)COOH, wherein m is an integer ranging from 2to 12. When the complementary functionality is a hydroxyl group, thepreferred linking group is a protected phosphoramidite. When thecomplementary functionality is sulfhydryl, the linking group can be amaleimide, halo acetyl, or iodoacetamide for example. See R. 35 Haugland(1992) Molecular Probes Handbook of Fluorescent Probes and ResearchChemicals, Molecular Probes, Inc., disclosing numerous modes forconjugating a variety of dyes to a variety of compounds which sectionsare incorporated herein by reference.

The invention also is directed to nucleic acid compositions containingdye pairs, which include one of the disclosed quencher compositions anda fluorescent dye that fluorescences on exposure to light of theappropriate wavelength. Suitable fluorescent dyes in the dye pair arethose that emit fluorescence that can be quenched by the quencher of thedye pair. In certain embodiments, the dye pair can be attached to asingle compound, such as an oligonucleotide. In other embodiments, thefluorescent reporter dye and the quencher can be on different molecules.

A wide variety of reactive fluorescent reporter dyes are known in theliterature and can be used so long as they are quenched by thecorresponding quencher dye of the invention. Typically, the fluorophoreis an aromatic or heteroaromatic compound and can be a pyrene,anthracene, naphthalene, acridine, stilbene, indole, benzindole,oxazole, thiazole, benzothiazole, cyanine, carbocyanine, salicylate,anthranilate, coumarin, fluoroscein, rhodamine or other like compound.Suitable fluorescent reporters include xanthene dyes, such asfluorescein or rhodamine dyes, including 6-carboxyfluorescein (FAM),2′7′-dimethoxy-4′5′-dichloro-6-carboxyfluorescein (JOE),tetrachlorofluorescein (TET), 6-carboxyrhodamine (R6G), N,N,N;N′-tetramethyl-6-carboxyrhodamine (TAMRA), 6-carboxy-X-rhodamine (ROX).Suitable fluorescent reporters also include the naphthylamine dyes thathave an amino group in the alpha or beta position. For example,naphthylamino compounds include 1-dimethylaminonaphthyl-5-sulfonate,1-anilino-8-naphthalene sulfonate and 2-p-toluidinyl-6-naphthalenesulfonate, 5-(2′-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS).Other fluorescent reporter dyes include coumarins, such as3-phenyl-7-isocyanatocoumarin; acridines, such as9-isothiocyanatoacridine and acridine orange;N-(p-(2-benzoxazolyl)phenyl)maleimide; cyanines, such asindodicarbocyanine 3 (Cy3), indodicarbocyanine 5 (Cy5),indodicarbocyanine 5.5 (Cy5.5),3-(-carboxy-pentyl)-3′-ethyl-5,5′-dimethyloxacarbocyanine (CyA);1H,5H,11H,15H-Xantheno[2,3,4-ij:5,6,7-i′j′]diquinolizin-18-ium, 9-[2(or4)-[[[6-[2,5-dioxo-1-pyrrolidinyl)oxy]-6-oxohexyl]amino]sulfonyl]-4 (or2)-sulfophenyl]-2,3,6,7,12,13,16,17-octahydro-inner salt (TR or TexasRed); BODIPY™ dyes; benzoxaazoles; stilbenes; pyrenes; and the like. Thefluorescent emission of certain reporter dyes is provided below.

Fluorophore Emission Max 6-Carboxyfluorescein (6-FAM) 520 nmTetrachlorofluorescein (TET) 536 nm Hexachlorofluorescein (HEX) 556 nmCy3 570 nm Tetramethylrhodamine (Tamra) 580 nm Cy3.5 596 nmCarboxy-x-rhodamine (Rox) 605 nm Texas Red 610 run Cy5 667 nm Cy5.5 694nm

The quencher of Example 2 is capable of absorbing the fluorescent energyin the range of about 500 to about 620 nm and therefore can be used toquench the fluorescence of fluorescein through Texas Red.

Many suitable forms of these fluorescent reporter dyes are available andcan be used depending on the circumstances. With xanthene compounds,substituents can be attached to xanthene rings for bonding with variousreagents, such as for bonding to oligonucleotides. For fluorescein andrhodamine dyes, appropriate linking methodologies for attachment tooligonucleotides have also been described. See for example, Khanna etal. U.S. Pat. No. 4,439,356; Marshall (1975) Histochemical J.,7:299-303; Menchen et al., U.S. Pat. No. 5,188,934; Menchen et al.,European Patent Application No. 87310256.0; and Bergot et al.,International Application PCT/U590/05565).

Preferably, when the dye pair is in a configuration in which thereporter dye is effectively quenched by the quencher dye, itsfluorescence is reduced by at least a factor of 80%, and more preferablyby 90%, 95%, or 98%, when compared to its fluorescence in the absence ofquenching. Compositions with 99%, 99.5%, 99.9% and higher levels ofquenching have also been prepared with the quencher of Example 2. Highlevels of quenching allow for the preparation of oligonucleotide probeshaving a high signal to noise ratio which is defined as the amount ofsignal present when the composition is in its maximal unquenched state(signal) versus its maximally quenched state (noise).

Probes having a high signal to noise ratio are desirable for thedevelopment of highly sensitive assays. To measure signal to noiseratios relative fluorescence is measured in a configuration where thequencher and fluorophore are within the Förster distance and thefluorophore is maximally quenched (background fluorescence or “noise”)and compared with the fluorescence measured when fluorophore andquencher are separated in the absence of quenching (“signal”). Thesignal to noise ratio of a dye pair of the invention will generally beat least about 2:1 but generally is higher. Signal to noise ratios ofabout 5:1, 10:1, 20:1, 40:1 and 50:1 are preferred. Ratios of 60:1, 70:1and even up to 100:1 and higher can also be obtained in some cases.Intermediate signal to noise ratios are also contemplated.

Suitable dye-pairs can be used in many configurations. For example, thedye pair can be placed on nucleic acid oligomers and polymers. In thisformat, a dye-pair can be placed on an oligomer having a hairpinstructure such that the fluorophore and quencher are within the Försterdistance and FRET occurs.

In other embodiments, dye pairs can be placed on an oligomer that canadopt a random coil conformation, such that fluorescence is quencheduntil the oligonucleotide adopts an extended conformation, as when itbecomes part of a duplex nucleic acid polymer. In general, theindividual dye moieties can be placed at any position of the nucleicacid depending upon the requirements of use.

Nucleic acid oligomers and polymers that include the dye pairs of theinvention can be used to detect target nucleic acids. In one method, theindividual components of a dye-pair can be on opposing, annealable,self-complementary segments of a single oligonucleotide such that whenthe oligonucleotide anneals to itself in the absence of exogenoussequences, FRET occurs. The oligonucleotide is constructed in such a waythat the internal annealing is disrupted and fluorescence can beobserved when it hybridizes to nucleic acid polymers having sufficientcomplementarity. Such an oligonucleotide can be used to rapidly detectnucleic acid polymers having sequences that bind to the oligonucleotide.In another embodiment, such a composition comprises two biomolecules,such as oligonucleotides, one of which is attached to a reporter dye andthe other of which is attached as a quencher dye.

Oligonucleotide probes lacking self-complementarity can also be utilizedin a similar manner. For example, a quencher and fluorophore can beplaced on an oligonucleotide that lacks the self-annealing property suchthat the random-coil conformation of the oligonucleotide keeps thefluorophore and quencher within a suitable distance for fluorescencequenching. Such oligonucleotides can be designed so that when theyanneal to desired target nucleic acid polymers the fluorophore andquencher are more separated and the spectral characteristics of thefluorophore become more apparent.

Other DNA binding formats are also possible. For example, twooligonucleotides can be designed such that they can anneal adjacent toeach other on a contiguous length of a nucleic acid polymer. The twoprobes can be designed such that when they are annealed to such anucleic acid polymer a quencher on one of the oligonucleotides is withina sufficient proximity to a fluorophore on the other oligonucleotide forFRET to occur. Binding of the oligonucleotides to the nucleic acidpolymer can be followed as a decrease in the fluorescence of thefluorophore.

Alternatively, a set of oligonucleotides that anneal to each other canbe configured such that a quencher and a fluorophore are positionedwithin the Förster distance on opposing oligonucleotides. Incubation ofsuch an oligonucleotide duplex with a nucleic acid polymer that competesfor binding of one or both of the oligonucleotides would cause a netseparation of the oligonucleotide duplex leading to an increase in thefluorescent signal of the reporter dye. To favor binding to the polymerstrands, one of the oligonucleotides could be longer or mismatches couldbe incorporated within the oligonucleotide duplex.

These assay formats can easily be extended to multi-reporter systemsthat have mixtures of oligonucleotides in which each oligonucleotide hasa fluorophore with a distinct spectrally resolvable emission spectrum.The binding of individual oligonucleotides can then be detected bydetermining the fluorescent wavelengths that are emitted from a sample.Such multi-reporter systems can be used to analyze multiplehybridization events in a single assay.

Oligonucleotides can also be configured with the disclosed quencherssuch that they can be used to monitor the progress of PCR reactionswithout manipulating the PCR reaction mixture (i.e., in a closed tubeformat). The assay utilizes an oligonucleotide that is labeled with afluorophore and a quencher in a configuration such that fluorescence issubstantially quenched. The oligonucleotide is designed to havesufficient complementarity to a region of the amplified nucleic acid sothat it will specifically hybridize to the amplified product. Thehybridized oligonucleotide is degraded by the exonuclease activity ofTaq polymerase in the subsequent round of DNA synthesis. Theoligonucleotide is designed such that as the oligomer is degraded, oneof the members of the dye-pair is released and fluorescence from thefluorophore can be observed. An increase in fluorescence intensity ofthe sample indicates the accumulation of amplified product.

Ribonucleic acid polymers can also be configured with fluorophores andquenchers and used to detect RNase. For example, a dye-pair can bepositioned on opposite sides of an RNase cleavage site in an RNasesubstrate such that the fluorescence of the fluorophore is quenched.Suitable substrates include nucleic acid molecules that have asingle-stranded region that can be cleaved and that have at least oneinternucleotide linkage immediately 3′ to an adenosine residue, at leastone internucleotide linkage immediately 3′ to a cytosine residue, atleast one internucleotide linkage immediately 3′ to a guanosine residueand at least one internucleotide linkage next to a uridine residue andoptionally can lack a deoxyribonuclease-cleavable internucleotidelinkage. To conduct the assay, the substrate can be incubated with atest sample for a time sufficient for cleavage of the substrate by aribonuclease enzyme, if present in the sample. The substrate can be asingle-stranded nucleic acid molecule containing at least oneribonucleotide residue at an internal position. Upon cleavage of theinternal ribonucleotide residue, the fluorescence of the reporter dye,whose emission was quenched by the quencher, becomes detectable. Theappearance of fluorescence indicates that a ribonuclease cleavage eventhas occurred, and, therefore, the sample contains ribonuclease activity.This test can be adapted to quantitate the level of ribonucleaseactivity by incubating the substrate with control samples containingknown amounts of ribonuclease, measuring the signal that is obtainedafter a suitable length of time, and comparing the signals with thesignal obtained in the test sample.

Generally, any of the described assays could be conducted with positivecontrols to indicate proper function of the assay.

The invention also provides kits that include in one or more containers,at least one of the disclosed quenching dye compositions andinstructions for its use. Such kits can be useful for practicing thedescribed methods or to provide materials for synthesis of thecompositions as described. Additional components can be included in thekit depending on the needs of a particular method. For example, wherethe kit is directed to measuring the progress of PCR reactions, it caninclude a DNA polymerase. Where a kit is intended for the practice ofthe RNase detection assays, RNase-free water could be included. Kits canalso contain negative and/or positive controls and buffers.

The following examples further illustrate the invention but, of course,should not be construed as in any way limiting its scope. In particularthe following examples demonstrate synthetic methods for obtaining thecompounds of the invention. Starting materials useful for preparing thecompounds of the invention and intermediates thereof, are commerciallyavailable or can be prepared from commercially available materials usingknown synthetic methods and reagents. All oligonucleotide sequences arewritten from the 5′-terminus on the left to the 3′-terminus on theright.

Example 1

This example demonstrates the chemical synthesis of the compound ofFormula 9. The abbreviation “CEP” in the formula stands forcyanoethylphosphoramidite.

The synthesis was as shown in Scheme 1 below. Cold concentrated HCl(4.25 mL) was added to a suspension of 4-nitro-1-naphthylamine (0.5 g,2.66 mmol) in water (1.6 mL) at 0° C. A solution of NaNO₂ (0.4 g) inwater (1 mL) was added dropwise at 0° C. over 15 min and the4-nitro-1-naphthylamine dissolved upon stirring. The solution wasallowed to react for 10 min and then urea (0.16 g) was slowly added.Next, a solution of N-ethyl-N-hydroxyethylaniline (0.45 g) in aceticacid (2.18 mL) was mixed into the solution. After 15 mins, sodiumacetate (5.31 g) in water (13.3 mL) was added slowly. The reactionmixture was stirred for 1 h at room temperature and the aqueous layerwas then extracted twice with ethyl acetate. The organic layer was driedover Na₂SO₄ and subjected to flash chromatography with ethylacetate togive the azo alcohol (2) as a dark oil (0.5 g, 52% yield). MS (FAB+)[M+]: calc'd for C₂₀H₂₀N₄O₃, m/z 364.4; observed, m/z 365.

N,N-diisopropylamino cyanoethyl phosphonamidic chloride (0.46 mL, 2.06mmol) was added dropwise at 0° C. to a solution of the azo alcohol (2)(0.5 g, 1.37 mmol) and triethylamine (0.38 mL, 2.74 mmol) in THF (20mL). The mixture was stirred at room temperature for 2 h. CH₂Cl₂ waspoured into the reaction mixtures and the organic layer washed twicewith water and dried over Na₂SO₄. Flash chromatography on silica withthe solvent EtOAc/PE/TEA: 20/70/10-40/50/10 gave 1 g of a dark oilrepresenting nearly quantitative recovery. Analysis of the product bysilica thin layer chromatography gave a product having an Rf of 0.49 inEtOAc/PE/TEA: 40/50/10. MS (FAB+) [M+]: calc'd for C₂₉H₃₇N₆O₄P, m/z564.2; observed, m/z 565.

The resulting phosphoramidite compound was used to prepareoligonucleotides containing the quencher moiety attached to the5′-terminal hydroxyl group. The phosphoramidite quencher was added tothe oligonucleotide during synthesis in the last addition reaction usingstandard automated phosphoramidate chemistry. Subsequently, theoligonucleotides were cleaved from the support, deprotected and purifiedusing standard methods.

Example 2

This example demonstrates the synthesis of a controlled pore glass (CPG)support for oligonucleotide synthesis that is derivatized with aquencher of the present invention. The synthetic method is shown belowin Schemes 2 and 3.

Mono-DMT-phenyl diethanolamine: A solution of 10 g of phenyldiethanolamine in 100 mL of pyridine was mixed for 3-4 h at roomtemperature with a solution of 6 g dimethoxytrityl-chloride (DMT-Cl) in150 mL of a 98:2 dichloromethane/pyridine solution. The reaction mixturewas concentrated to dryness under vacuum. The residue was dissolved in200 mL of ethyl acetate, washed with two portions of 100 mL of deionizedwater, and the organic layer dried over Na₂SO₄. The organic solution wasconcentrated and purified by column chromatography using a 300 g ofsilica gel column developed with 30/65/5 ethylacetate/hexanes/triethylamine to yield 5.25 g (20% yield) ofmono-DMT-phenyl diethanolamine TLC: R_(f) 0.55(EtAc/hexanes/Et₃N—40/55/5). ¹H NMR (CDCl₃) δ 7.38 (d, J=8 Hz, 2H), 7.27(d, J=8 Hz, 4H), 7.38 (d, J=8 Hz, 2H), 7.24-7.12 (m, 6H), 6.76 (d, J=8Hz, 4H), 6.66 (d, J=8 Hz, 2H), 3.74 (s, 6H), 3.74 (t, J=7.5 Hz, 2H),3.54 (t, J=7.5 Hz, 2H), 3.51 (t, J=7.5 Hz, 2H), 3.33 (t, J=7.5 Hz, 2H),2.23 (br. s, 1H).

Mono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline (2): Coldconcentrated HCl (17 mL) was added dropwise at 0° C. over 15 min to asuspension of 4-nitro-1-naphthylamine (2 g) in cold water (6 mL) at 0°C. Then NaNO₂ (1.6 g) in cold water (4 mL) was added dropwise at 0° C.over 15 min and the 4-nitro-1-naphthylamine dissolved upon stirring.LiBF₄ (1.38 g) in H₂O (3 mL) was added dropwise at 0° C. The reactionmixture was stirred at 0° C. for 30 min. A brownish yellow powder (3.08g) of naphthyl-1-nitro-4-tetrafluoroborate diazonium salt (1) wasobtained after filtering and rinsing the solution with cold water,methanol, and ether. A solution of 4 g of mono-DMT-phenyl diethanolaminein 50 mL of dimethylsulfoxide (DMSO) was added with stirring at 10-15°C. over 10-15 min to a chilled solution of 2.8 g of diazonium salt (1)in 50 mL of DMSO at 10-15° C. in a water bath. After an additional 15min of stirring, 3 mL of triethylamine was added to the reaction mixturefollowed by 100 mL of ethyl acetate. The reaction mixture was washedwith 3×30 mL of deionized water and the organic layer was dried overNa₂SO₄. The solvent was removed and product was purified by columnchromatography with 300 g of silica gel to provide 1.8 g ofmono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline (2). TLC: R_(f)0.65 (DCM/Et₃N—80/20). ¹H NMR (CDCl₃) δ 9.04 (d, J=8.4 Hz, 1H), 8.68 (d,J=8.4 Hz, 1H), 8.34 (d, J=8.4 Hz, 1H), 7.96 (d, J=9.2 Hz, 2H), 7.81-7.71(m, 3H), 7.39 (d, J=8 Hz, 2H), 7.27 (d, J=8 Hz, 4H), 7.24-7.19 (m, 3H),6.78 (d, J=8 Hz, 4H), 6.77 (d, J=8 Hz, 2H), 3.88 (t, J=7.5 Hz, 2H), 3.75(s, 6H), 3.78-368 (m, 4H), 3.47 (t, J=7.5 Hz, 2H), 1.57 (br. s, 1H).

Mono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline phosphoramidite(3): A solution of 0.2 ml ofN,N-diisopropylamino-cyanoethyl-phosphoramidochloride was stirred into asolution of 0.3 g of alcohol (2) in 20 mL of anhydrous THF and 1 mL oftriethylamine for 5 min at 0-5° C. After 15 min of additional stirringthe reaction mixture was warmed to room temperature. The solvent wasevaporated under a vacuum and the residue purified by columnchromatography through 50 g of silica gel (EtOAc/PE/TEA:10/85/5-40/55/5). TLC: R_(f) 0.65 (EtOAc/PE/Et₃N—40/55/5). ¹H NMR(CDCl₃) δ 9.05 (d, J=8.4 Hz, 1H), 8.68 (d, J=8.4 Hz, 1H), 8.34 (d, J=8.4Hz, 1H), 7.96 (d, J=9.2 Hz, 2H), 7.81-7.71 (m, 3H), 7.39 (d, J=8 Hz,2H), 7.27 (d, J=8 Hz, 4H), 7.24-7.19 (m, 3H), 6.78 (d, J=8 Hz, 4H), 6.76(d, J=8 Hz, 2H), 3.85-3.75 (m, J=7.5 Hz, 4H), 3.76 (s, 6H), 3.70 (t,J=7.5 Hz, 2H), 3.41 (t, J=7.5 Hz, 2H), 2.58 (t, J=8.0 Hz, 2H), 1.20 (s,3H), 1.18 (s, 3H), 1.17 (s, 3H), 1.15 (s, 3H). ³¹P NMR δ 148.39.

Mono-DMT-4-(1-nitro-4-naphthylazo)-N,N-diethanolaniline-CPG 4: One gramof 3′-phosphate CPG (see Formula 10), was treated with 3×10 mL of 3%(v/v) dichloroacetic acid in dichloromethane (DCM) and then washed with5×10 ml of acetonitrile. A solution of 0.4 g of phosphoramidite (3) in 5mL of dry acetonitrile and 5 mL of 0.45 Methylthiotetrazole was added toCPG using an argon sparge. After 20 min the reaction mixture wasremoved, and the derivatized CPG was washed with 5×10 mL ofacetonitrile. A 10 mL solution of 10% (v/v) acetic anhydride solution inTHF and 10 mL of 10% (v/v) methylimidazole in 8:1 mixture ofTHF/pyridine were added to the resulting derivatized CPG using an Argonsparge. After 30 min the reaction mixture was removed and thederivatized CPG was washed with 5×10 mL of acetonitrile. A 10 mLsolution of 0.02 M iodine in THF/pyridine/H₂O solution was added to thederivatized CPG for 5 min. Then the derivatized CPG was removed, andwashed with 5×10 mL of acetonitrile and dried overnight invacuum-desiccator to obtain 1.1 g of derivatized CPG (4).

The derivatized support (4) was used to support oligonucleotidesynthesis using standard oligonucleotide synthetic techniques. DMT groupremoval, nucleotide additions, and deprotection of the full lengtholigonucleotide were all carried out by standard methods.Oligonucleotides synthesized on this support had the quencher positionedat their 3′-terminal hydroxyl group.

Example 3

This example demonstrates the signal to noise ratio (S:N) ratio ofoligonucleotides containing both fluorescein and the azo quencher asprepared in Examples 1 and 2. The method involved measuring the relativefluorescence of the oligonucleotide while it was in a native singlestranded configuration (background fluorescence or “noise”) andcomparing that with the fluorescence of the oligonucleotide measuredwhen fluorophore and quencher are more separated (“signal”), as when theoligonucleotide is cleaved to separate the azo-quencher moiety from thefluorophore.

Oligonucleotide Synthesis. Dual-labeled oligonucleotides were made withthe azo-quencher at either the 5′-end or 3′-end of the molecule and withthe fluorescein reporter group placed at the opposing end (6-FAM, singleisomer 6-carboxyfluorescein, Glen Research, Sterling, Va.). Forcomparison, an oligonucleotide was made that incorporated a commerciallyavailable quenching group, the Black Hole Quencher™-2 group (BiosearchTechnologies, Novato, Calif., hereinafter, BHQ_(—)2). Theoligonucleotides were purified by HPLC using standard methods. Theoligonucleotides are shown below and are written with their 5′-terminito the left. The oligonucleotides were synthesized using standardautomated phosphoramidite synthetic methods. For synthesis of SEQ ID No.1, the Azo-Quencher prepared in Example 1 was added to theoligonucleotide in the last addition reaction as if it were anothernucleotide addition. For synthesis of SEQ ID No. 2, the Azo-Quencherderivatized CPG described in Example 2 was used and nucleotides wereadded using standard automated methods.

Probe Sequence SEQ ID No. 1 (AzoQuencher)-ACCCGTTCACCCTCCCCCAGT- (6-FAM)SEQ ID No. 2 (6-FAM)-ACCCGTTCACCCTCCCCCAGT- (AzoQuencher) SEQ ID No. 3(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(BHQ_2)

The structure of the oligonucleotide linkage to the 3′-AzoQuencher groupin oligonucleotide SEQ ID No. 2 is shown below in Formula 11. Thelinkage of the AzoQuencher to the oligonucleotide is through the3′-hydroxyl group of the oligonucleotide.

The structure of the oligonucleotide linkage to the 5′-AzoQuencher groupin oligonucleotide SEQ ID No. 1 is shown below in Formula 12. Thelinkage of the AzoQuencher to the oligonucleotide is through the5′-hydroxyl group of the oligonucleotide.

Signal to Noise (S:N) Assay of Fluorescence-Quenched Linear Probes.Oligonucleotides were evaluated for 6-Fam quenching efficiency using thefollowing protocol. Probe oligonucleotides (SEQ ID Nos. 1-3) wereindividually resuspended at 100 μM concentration in HPLC-grade water.From this stock solution, 2 mL of 100 nM probe solution was preparedwith “STNR” Buffer, comprising 10 mM Tris pH 8.3, 50 mM KCl, 5 mM MgCl₂,1 mM CaCl₂ which was split into two identical 1 mL fractions. Onefraction was retained without enzyme treatment as the backgroundcontrol. The second fraction was subjected to nuclease degradation.Nuclease degradation was with micrococcal nuclease, 15 units (Roche, 15U/μL) which was mixed into the oligonucleotide solution and incubated at37° C. for 1 h. The relative fluorescence intensity of each sample wasmeasured with a PTI QuantaMaster Model C-60 cuvette-basedspectrofluorometer (Photon Technology International, Monmouth Jct., NJ).The fluorescence of the solution containing undegraded (without nucleaseenzyme treatment) probe was considered to be “background” or “noise.”The fluorescence measurement of the solution containing degraded probe(with nuclease treatment) was treated as “signal.”

The signal to noise ratios (S:N) were calculated and are shown below inTable 1.

TABLE 1 Dye/Quencher RFU RFU S:N Probe ID 5′-3′ Background Signal RatioSEQ ID No. 1 AzoQuencher-6FAM 4.20E+05 6.76E+06 16 SEQ ID No. 26FAM-AzoQuencher 7.36E+04 2.32E+06 31 SEQ ID No. 3 6FAM-BHQ2 2.14E+055.43E+06 25 RFU = relative fluorescence units

As shown in Table 1 the signal to noise ratios (S:N Ratio) of SEQ IDNos. 1-3 are all substantial and comparable, demonstrating that thenovel azo-quencher of this invention is capable of quenching afluorescein with similar or better efficiency than a commonly employedcommercially available quencher group. The azo-quencher will also beeffective at quenching a variety of other fluorescent reporter dyes,including tetrachlorofluorescein, hexachlorofluorescein and Texas Red.

Example 4

Fluorescence-quenched probes are frequently used to detect targetnucleic acid sequences using amplification reactions, such as thepolymerase chain reaction (PCR) (Mullis et. al., 1986). This exampledemonstrates the use of fluorescent probes that contain the Azo-Quencherof this invention to detect PCR amplified DNA.

Oligonucleotide primers and probes were synthesized as described inExample 3. Primers, probes, and target nucleic acids were as shown inTable 2 below. Probes used were SEQ ID No. 4-6. Primers used are SEQ IDNo. 7 and 8. The target nucleic acid is SEQ ID No. 9, a 220 basepair(bp) amplicon derived from the murine bHLH protein Ptfl-p48 gene(Genbank #AF298116), cloned into the pCRII-TOPO vector (Invitrogen,Carlsbad, Calif.), and is hereafter referred to as the “p48-genetarget”.

TABLE 2 Probes: SEQ ID No. 4 (AzoQuencher)-ACCCGTTCACCCTCCCCCAGT-(6-FAM)SEQ ID No. 5 (6-FAM)-ACCCGTTCACCCTCCCCCAGT-(AzoQuencher) SEQ ID No. 6(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(BHQ2) Forward Primer: MP48 F968SEQ ID No. 7 CAGAAGGTTATATCTGCCATCG Reverse Primer: MP48 R1187SEQ ID No. 8 CTCAAAGGGTGGTTCGTTCTCT Target Nucleic Acid SequenceForward Primer                 Probe SEQ ID No. 9CAGAAGGTTATCATCTGCCATCGAGGCACCCGTTCACCCTCCCCCAGTGACCCGGATTATGGTCTCCCTCCTCTTGCAGGGCACTCTCTTTCCTGGACTGATGAAAAACAGCTCAAAGAACAAAATATCATCCGTACAGCTAAAGTGTGGACCCCAGAGGACCCCAGAAAACTCAACAGTCAAATCTTTCGACAACATA GAGAACGAACCACCCTTTGAG    Reverse Primer

PCR amplification was done with the Stratagene (La Jolla, Calif.)Brilliant Plus™ Quantitative PCR core Reagent Kit according to themanufacturer's directions. Reactions were carried out in a 25 μL volumewith 200 nM each of the amplification primers and fluorescent quenchedprobe and 5000 copies of target DNA. Cycling conditions were 50° C. for2 min, 95° C. for 10 min, then 40 cycles of 2-step PCR with 95° C. for15 sec and 60° C. for 1 min. An ABI Prism™ 7000 Sequence Detector(Applied Biosystems Inc., Foster City, Calif.) was used for PCR andfluorescence measurements. All assays were performed in triplicate.Results for different probes are presented in Table 3 below. In Table 3,the cycle threshold (Ct) value is defined as the cycle at which astatistically significant increase in fluorescence is detected abovebackground. A lower Ct value is indicative of a higher concentration oftarget DNA. Table 3 shows that assays with each oligonucleotide probehad similar Ct values and therefore functioned similarly in theseassays.

TABLE 3 Probes: Sequence Avg. Ct SEQ ID No. 4(AzoQuencher)-ACCCGTTCACCCTCCCCCAGT-(6-FAM) 24.3 SEQ ID No. 5(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(AzoQuencher) 23.6 SEQ ID No. 6(6-FAM)-ACCCGTTCACCCTCCCCCAGT-(BHQ2) 23.2

Relative fluorescence levels collected during PCR for each probe weregraphically plotted against cycle number and are shown in FIG. 1. Thisexample demonstrates that probe compositions comprising the newazo-quenchers of the invention perform well in a quantitative real-timePCR assay and are functionally equivalent to probes that contain otherquencher moieties.

Example 5

This example shows an absorbance spectrum of an oligonucleotide modifiedat its 5′ terminus to contain the AzoQuencher of Formula 8 in Example 1.The oligonucleotide was made using standard automated phosphoramiditenucleotide synthetic methods where the last addition cycle was carriedout with the molecule of Formula 8. The composition of theoligonucleotide is shown below.

(Azo-Quencher)-CAGAGTACCTGA SEQ ID No. 10

Once synthesized, the oligonucleotide was suspended in HPLC-grade waterat 400 nM concentration. Optical absorbance was measured in 10 mM TrispH 8.0, 1 mM EDTA (TE buffer) with a sub-micro quartz cuvette with 1-cmpath length in a Hewlett Packard Model 8453 spectrophotometer (HewlettPackard, Palo Alto, Calif.). Absorbance density was recorded from 220 nmto 700 nm and is shown in FIG. 2.

As shown in FIG. 2, the absorbance spectrum is broad, ranging from 420to 620 nm, with peak absorbance at 531 nm. This absorbance rangeoverlaps with the fluorescence emission of a wide variety offluorophores commonly used in molecular biology applications. For FRETbased quenching mechanisms, this spectrum is positioned to offer maximumquenching capacity for dyes in the spectral range of fluorescein.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

1. A method for detecting hybridization of nucleic acid polymerscomprising: a) incubating a first nucleic acid polymer complimentary toa second nucleic acid polymer having, attached thereto, a quenchercompound of Formula 1:

wherein R₁₋₆ on the conjugated ring system are individually an electronwithdrawing group, alkyl group, aryl group, hydrogen, heteroaryl group,or a five or six member ring structure formed from the R₁, R₂ pair, theR₃, R₄ pair, the R₄, R₅ pair, or the R₅, R₆ pair; and wherein R₇ is anunsubstituted or substituted aryl or heteroaryl group, the aryl orheteroaryl group being selected from the group consisting of phenyl,xylyl, tolyl, pyridyl, and anilyl groups, with the proviso that the arylgroup is not an unsubstituted phenyl or tolyl, and wherein the anilylgroup is a group of Formula 2:

wherein L and L′ are the same or different and are a C₁₋₁₀ group,—C₁₋₁₀—O—R₈, or —C₁₋₁₀—O—R₉, wherein C₁₋₁₀ is a C₁₋₁₀ alkyl group, andwherein R₈ and R₉ are independently a hydrogen, trityl, alkoxytrityl,silyl, cyanoethylphosphoramidite, or phosphoramidite with the provisothat L and L′ are not both methyl, and when L or L′ is ethyl, then theother is not hydroxyethyl, and wherein at least one of the first andsecond nucleic acid polymers comprises a fluorophore and fluorescencefrom the fluorophore can be quenched by the quencher compound of Formula1; and b) measuring the fluorescence such that when the first and secondnucleic acid polymers are hybridized, the fluorescence of thefluorophore is reduced.
 2. The method of claim 1 further comprisingaltering the spatial relationship between the fluorophore and quencher.3. The method of claim 2 wherein the altering of the spatialrelationship between the fluorophore and quencher is a result of thehybridization of the first and second nucleic acid polymers.
 4. Themethod of claim 1 wherein the fluorophore and quencher composition arelinked to a single nucleic acid polymer.
 5. The method of claim 1further comprising, when the first and second nucleic acid polymers arehybridized to each other, releasing the fluorophore or quenchercomposition from the hybridized structure.
 6. A method for synthesizingan oligonucleotide having attached thereto a fluorescent quencher ofFormula 1:

wherein R₁₋₆ on the conjugated ring system are individually an electronwithdrawing group, alkyl group, aryl group, hydrogen, heteroaryl group,or a five or six member ring structure formed from the R₁, R₂ pair, theR₃, R₄ pair, the R₄, R₅ pair, or the R₅, R₆ pair; and wherein R₇ is anunsubstituted or substituted aryl or heteroaryl group, the aryl orheteroaryl group being selected from the group consisting of phenyl,xylyl, tolyl, pyridyl, and anilyl groups, with the proviso that the arylgroup is not an unsubstituted phenyl or tolyl, and wherein the anilylgroup is a group of Formula 2:

wherein L and L′ are the same or different and are a C₁₋₁₀ group,—C₁₋₁₀—O—R₈, or —C₁₋₁₀—O—R₉, wherein C₁₋₁₀ is a C₁₋₁₀ alkyl group, andwherein R₈ and R₉ are independently a hydrogen, trityl, alkoxytrityl,silyl, cyanoethylphosphoramidite, or phosphoramidite with the provisothat L and L′ are not both methyl, and when L or L′ is ethyl, then theother is not hydroxyethyl, the method comprising: obtaining a firstcompound of Formula 5:

treating the primary amino group of the first compound in the presenceof NaNO2, followed by LiBF4, to form an diazonium salt; reacting thediazonium salt with a second compound having a substituted aryl ring toform a product having an azo group; covalently incorporating the productinto an oligonucleotide.